ABCA1: The current model for reverse cholesterol transport (RCT) proposes that High-Density Lipoproteins (HDL) transports excess cholesterol (C) derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL-C. To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-AdV) that targets expression of mouse ABCA1 in vivo to the liver. Compared to mice injected with control AdV, infusion of rABCA1-AdV into C57Bl/6 and ABCA1-KO mice resulted in a significant expression of ABCA1 mRNA in the liver. ApoA-I dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated four days after rABCA1-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 and ABCA1-KO mice raised baseline levels of TC, PL, FC, HDL-C, apoE and apoA-I by 150-300% (p<0.05 all) and in ABCA1-KO mice led to a major shift from lipid-poor to mature HDL. ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMGCoA reductase (3.5-fold), LDLr (2.1-fold) and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C. ABCA1: ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL. FXR: FXR is the transcription factor that regulates the hepatic production, canalicular transport and intestinal reabsorption of bile acids (Cell 102:731-744). To address the physiological role of FXR on cholesterol homeostasis, we evaluated the major pathways of cholesterol metabolism in the FXR-ko mouse model. (1) On a chow diet, FXR-ko mice had increased plasma TC, CE and HDL-C compared to C57BL controls. By 2-D gel electrophoresis, pre-? and ?-HDL levels were increased in FXR-ko compared to controls despite similar hepatic and intestinal apoA-I, and apoA-II and ABCA1 mRNA expression. The catabolism of HDL I125- apoA-I and I131-apoA-II were also similar but the plasma decay of H3-CE labeled HDL was delayed in FXR-ko mice, consistent with reduced hepatic SR-BI protein expression. Surprisingly, the flow of hepatic biliary cholesterol determined after bile duct cannulation was increased in FXR-ko mice vs. controls despite decreased hepatic mRNA expression of the sterol transporters ABCG5 and ABCG8. On a 0.4% cholic acid diet, the flow of biliary cholesterol was dramatically reduced in FXR-ko mice. In contrast, the biliary cholesterol flow increased in control mice upon 0.4% CA feeding. (3) The intestinal absorption of dietary cholesterol was increased in FXR-ko mice compared to controls. In addition, FXR-ko mice had increased production of Lp-B after triton injection, n=4,p<0.02). In summary, these in vivo data demonstrate that FXR (i) is a positive regulator of plasma HDL metabolism, (ii) modulates the biliary secretion of cholesterol, and (iii) is a negative regulator of intestinal cholesterol absorption. We conclude that FXR plays a central role in the regulation of the entero-hepatic circulation of cholesterol.